This invention relates to novel heparitinases which degrade heparan sulfate or heparin, a process for preparing the same and bacteria producing the same.
Heparitinase is a group of enzymes which cleave glucosaminide linkages into heparan sulfate (hereinafter abbreviated to as "HS") and/or heparin (hereinafter abbreviated to as "Hep") which are heteropolysaccharide composed of repeating disaccharide units of N-acetyl-D-glucosamine and uronic acid as a basic structure, and are available as a reagent for analyzing HS or Hep in body or investigating these substances in vivo. Also, in recent years, it has attracted attention due to its availability as an agent used to prepare low molecular weight heparin which is now being developed as an antithrombic agent, or a material (Hep removing agent) for decreasing undesirable side effects of Hep which becomes a problem in therapy using an extracorporeal circulation apparatus. Thus, it can be expected to be used for various purposes for diagnosis and curing.
Heparitinase which can be used for the above use is required to have various properties such as different substrate specificity which recognizes the difference in saccharide chain structure such as the presence or absence, and a bonded position of a sulfate group, and it is desired to be supplied as an enzyme source stably with a large amount. Beginning from this point, the inventors ask for researched on an enzyme which satisfies the above requirements from microorganism origin, investigated bacteria which produce said enzyme and already found three kinds of heparitinases from bacteria belonging to Flavobacterium (Japanese Provisional Patent Publication No. 57183/1990).
As a report referred to in detail purification and properties of heparitinase which is microorganism origin, there have been known, in addition to the above, enzymes isolated from bacteria belonging to Flavobacterium or Bacillus, and disclosed in, for example, Summary of 10th International Glycoconjugate Symposium (p. 330, 1989) or Japanese Provisional Patent Publication No. 142470/1990. These known enzymes are available for the above objects, but it has been further demanded to obtain a novel heparitinase having different substrate specificity in order to accomplish the above objects.